阻止癌症轉移被列入治療癌症中重要的一環。過去，我們發現懸浮癌細胞表面組裝之Fibronectin (FN) matrix與肺臟微血管內皮細胞上Dipeptidyl peptidase IV (DPP IV)的結合在癌症轉移過程中扮演重要角色。藉由阻斷FN與DPP IV的結合或許是一種抑制癌症轉移的理想策略。基於此理由，我們定位出一段可與FN結合的DPP IV片段，DP4A，其二者結合的Kd (解離常數)為0.0267μM。雖然透過細胞黏著實驗確實發現DP4A對FN/DPP IV結合有很好的抑制作用，不過在in vivo，我們卻無法觀察到轉移有被抑制的現象。究其原因，發現只要在培養液中移除未與癌細胞結合的DP4A，細胞黏著的抑制現象就會消失。探討DP4A抑制能力消失的原因，源於癌細胞表面組裝之纖連蛋白基質與DP4A結合後會持續將DP4A吞噬並降解。文獻指出細胞表面之FN也有被吞噬降解的現象，或許DP4A結合上FN後一起被吞噬。此假設經由螢光染色共位標定兩者獲得證實。另一方面，我們觀察到吞噬後的FN與細胞內之EEA-1及LAMP-1共位，更進一步確認FN/DP4A被吞噬後走向lysosomal degradation途徑。由於我們的目的是利用DP4A作為抑制轉移的策略，因此阻斷吞噬與降解來保留DP4A仍在細胞表面上維持與FN基質的結合便成首要目標。一方面，從老鼠尾靜脈打入大量DP4A及乳癌細胞 (4T1)，使血液充滿DP4A以提供癌細胞表面之FN做結合，果然有明顯抑制小鼠癌細胞轉移的效果。我們將癌細胞施予DP4A，觀察其對細胞生長並無影響，更加確定DP4A並非藉由抑制細胞生長影響癌症轉移的能力。另一方面，單獨施予癌細胞PAO (吞噬抑制劑)及NH4Cl(降解抑制劑)並不影響其轉移能力，但能使更多DP4A留在細胞表面。未來計劃利用此二抑制劑阻斷吞噬與降解作用來達成DP4A抑制癌細胞轉移的目的。綜合以上成功的結果，DP4A可望被發展成為一種理想的抗癌症轉移藥物。

Metastatic inhibition is one of important strategies in cancer therapy. We found that cancer cell surface-associated (CSA) fibronectin (FN) mediates lung-specific cancer metastasis via interaction with lung endothelial Dipeptidyl peptidase IV (DPP IV). Identification of the FN-binding domain in DPP IV may help develop anti-adhesive metastasis-inhibiting drugs. For that purpose, we have located the FN-binding site in the N-terminal region of DPP IV and generated a recombinant peptide representing this site as DP4A. The Kd of DP4A for FN-binding was ~0.0267μM. While DP4A inhibited in vitro DPP IV/FN-mediated adhesion, no in vivo anti-metastatic effect was observed. Tremendous dilution of the free DP4A in the circulation may explain the failure of our in vivo data, in that DP4A lost its inhibitory effect once deprived of the free DP4A during the adhesion assay. We further demonstrated that the CSA FN-bound DP4A was soon endocytosed and degraded by cancer cells. Therefore, continuously provide free-DP4A may maintain its persistent cell surface-presence and the inhibitory effect. Next we examined the molecular mechanism of this endocytotic phenomenon with immunofluorescence staining and colocalization techniques and found that DP4A was endocytosed together with CSA FN, which has been reported to be endocytosed after binding to its cell surface receptor. Moreover, the endocytotic FN/DP4Acomplex was shown to be colocalized with the early endosome and lysosomal markers EEA-1and LAMP-1, respectively. Our results provide the evidence for the lysosomal degradation of endocytosed FN and DP4A. To improve the inhibitory effect of DP4A on cancer metastasis, we significantly increased the injected DP4A concentration, to which the adhesion inhibitory effect could be well maintained. Expectedly, the metastasis of cancer cells in the lungs was thus greatly inhibited. This metastatic inhibition was apparently not due to cell growth arrest. An alternative approach was attempted in which we blocked the endocytotic protein degradation by treating cancer cells with PAO (endocytosis inhibitor) or NH4Cl (degradation inhibitor). We first showed that these two inhibitors were able to keep the DP4A still bound to FN on cell surfaces from being endocytosed. We are currently testing these inhibitors in the in vivo metastasis assay to see whether they are able to enhance the inhibitory effect of DP4A on lung metastasis without providing excess free DP4A. Altogether, we provide a novel and potent anti-adhesive strategy in cancer metastasis prevention employing a FN-binding peptide of DPP IV in inhibiting the FN/DPP IV-mediated cancer metastasis.

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